The purpose of this project is a detailed investigation at the molecular level of the mechanism of action of vitamin D (calciferol) and its two primary metabolites 25-OH-vitamin D and 1,25-(OH)2-vitamin D. One of the hallmarks of vitamin D action is that there is a finite time lag between administration of the vitamin and the initiation of every one of its physiological responses. In view of this it has seemed appropriate to initiate a plan of attack that would place heavy emphasis on elucidating the nature of the biochemical and physiological events that occur between administration of the vitamin and initiation of the various physiological responses. Of necessity, this implies studying directly the biochemical fate of vitamin D and its metabolites. It will be particularly important to accurately determine the relative biological activity of the several vitamin D metabolites in the target tissues of intestine, bone and possibly kidney and to carry out an extensive examination and biochemical characterization of the subcellular receptors for these metabolites. Current evidence indicates that 1,25-(OH)2-vitamin D localizes in the nuclear chromatin fraction of the target intestinal mucosal cell and thereby initiates the physiological responses attributed to vitamin D. In addition, studies will be initiated to carry on both the enzymatic and organic synthesis of 1,25-dihydroxyvitamin D. The ready availability of this compound will greatly stimulate research into the fundamental action of vitamin D and further expand the clinical testing of this substance. It should then ultimately be possible to identify and characterize the effect of vitamin D in calcium transport system both in the whole cell and at the subcellular level.